Hyperammonemia is a metabolic disturbance characterized by excess ammonia in the blood. It is a dangerous condition that may lead to brain dysfunction (encephalopathy) and even death. Some cases of sudden infant death syndrome (SIDS) have been linked to undiagnosed metabolic disorders that caused hyperammonemia. A number of different conditions may cause hyperammonemia, such as inherited genetic mutations, liver damage, blind loop syndrome associated with gastrointestinal surgery, inflammatory bowel disease and some types of chemotherapy medications used for cancer treatment. Hyperammonemia is typically simple to treat if detected early enough. Unfortunately, there are no currently available, simple, quick tests for detecting hyperammonemia. This means that many cases of hyperammonemia go undetected until it is too late, often resulting in patient death.
Conditions that cause hyperammonemia may be inherited or acquired. Many different inherited genetic mutations may cause hyperammonemia. Ammonia is converted to the less toxic substance urea prior to excretion in urine by the kidneys. Some children have genetic mutations that disrupt the urea cycle in the body. These children require lifetime monitoring of blood ammonia levels. Acquired causes of hyperammonemia, including those mentioned above, also require long term monitoring of blood ammonia levels. Unfortunately, there is no convenient way for patients to monitor their blood ammonia levels on an ongoing basis.
Sudden infant death syndrome is the largest single cause of death in children in the industrialized world. A small but significant fraction of SIDS deaths are due to an unrecognized metabolic disorder associated with hyperammonemia. Many of these disorders become symptomatic either in the newborn or in the first few months of life. Indeed, some states perform neonatal tests for genetic diseases, such as fatty acid oxidation disorders or organic acidemias, which are associated with hyperammonemia. Rather than measuring blood ammonia levels directly, the tests employ tandem mass spectrometry to detect specific metabolites that accumulate in these conditions. The tests must be performed from a drop of blood obtained by a heel stick of the newborn infant, because venipuncture is technically difficult, morbid, and invasive. Currently, the United States screens for only a small number of hyperammonemia-associated defects. Other economically advanced countries offer few screening tests of any kind.
Although hyperammonemia damages the brain, it can be effectively and simply treated if detected early enough. One simple treatment involves administering lactulose to the patient. Lactulose is a non-absorbed sugar, which acidifies the stool and thus prevents absorption of ammonia into the blood from the intestine. Another treatment involves administering the antibiotic Rifaxamin, which kills urea-producing bacteria in the intestine. These treatments are simple, inexpensive and effective, if used early enough in the course of hyperammonemia.
The most pressing issue in detecting and treating hyperammonemia lies in detection. All of the currently available detection methods require processing blood to acquire plasma, and then testing the plasma for ammonia. Processing involves spinning the blood in a centrifuge to separate the plasma from the red blood cells. This must be done in a lab that has centrifuge equipment. The plasma must then be quickly tested, as soon as possible after blood processing. In most cases, the blood processing and ammonia level measurement must be carried out in two different areas of the hospital. Thus, a patient must go to a blood-processing lab attached to a hospital to have blood taken for the test. Additionally, the current tests for blood ammonia require an amount of blood that can only be acquired by venipuncture. This makes testing infants and small children additionally challenging.
The most commonly used clinical laboratory test for measuring plasma ammonia utilizes the glutamate dehydrogenase reaction, in which ammonium ion reacts with 2-oxoglutarate and NADPH to form glutamate, NADP and water. Absorbance spectroscopy at 340 nm measures the decrease in NADPH. The assay is highly specific and effective over a broad range of ammonia concentrations. However, the procedure is lengthy and complex, requiring venipuncture for several milliliters of blood, followed by centrifugation for plasma. Samples must be transported on ice to a central laboratory to minimize false elevation of ammonia levels from glutamine deamination in the blood cells. Thus, the standard clinical test is incompatible with close monitoring of patients at risk for hyperammonemia.
One alternative to the standard plasma ammonia analysis is to measure the ammonia concentration in whole blood. To avoid interference from other blood components, this strategy liberates ammonia in gaseous form for separate quantification. Gas-sensing electrodes detect ammonia by release of ammonia by alkalization of the whole blood sample. The electrode is placed above the surface of the sample and protected by a gas permeable membrane. This method has two major disadvantages, however—it requires a large amount of blood to work, and it takes a long time to analyze one sample—10 to 15 minutes at low ammonia levels.
Colorimetric reactions may also be used measure ammonia by spectrometry. Such reactions include the indophenol reaction, which generates a blue color, and the Nessler reaction, which generates a brown-orange color. The disadvantage of such methods is that other substances in the blood, such as amino acids and glutamine, can affect the reactions, leading to inaccuracy.
A more recent colorimetric method for measuring ammonia in whole blood has been developed and incorporated into a device known as the Blood Ammonia Checker or, in a more recent version, the PocketChem Blood Ammonia Analyzer (available from Woodley Equipment Company Ltd.). To measure ammonia, a small drop of blood is placed on a test strip that contains alkaline salts that liberate ammonia from the blood. The ammonia diffuses through a porous separator to a color-developing layer containing bromocresol green. The ammonia is quantified by colorimetry after 3-4 minutes. The problem with this device is that it has been shown to have problems with accuracy. Additionally, sensitivity for detecting elevated ammonia levels is questionable. The dynamic range of a dye-based assay is necessarily limited by the pKa of the dye and the sensitivity of the color detection system. Finally, the price of the Blood Ammonia Analyzer is $3,000.
Therefore, it would be very desirable to have an improved method and device for testing blood ammonia levels. Ideally, such a method and device would be simple to use, relatively inexpensive, and portable. Also ideally, the method and device would be convenient for use in a hospital, physician's office or patient's home. Finally, it would be ideal if such a method and device could be used for one-time blood ammonia level testing, ongoing monitoring of blood ammonia levels, or both. At least some of these objectives will be met by the embodiments described below.